p-Hydroxybenzoic acid (pHBA) is the major monomeric component (˜65% by weight) of Zenite™, a Liquid Crystal Polymer (LCP). LCP's have superior properties over conventional resins such as high strength/stiffness, low melt viscosity, excellent environmental resistance, property retention at elevated temperatures, and low gas permeability. However, current synthetic methods for the synthesis of pHBA (Kolbe-Schmitt reaction (Kolbe and Lautemann, Ann. 113:125 (1869)), are prohibitively expensive, and an inexpensive route to LCP monomers would open up many new applications for their use in the automotive, electrical, and other industries. Biological production offers one potential, less expensive route to pHBA production.
pHBA has been produced in microbial systems. For example, JP 06078780 teaches pHBA preparation by culturing benzoic acid in the presence of microorganisms (preferably Aspergillus) that oxidize benzoic acid to pHBA. Additionally, strains of Enterobacter with the ability to convert p-cresol to pHBA have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of Pseudomonas putida with the ability to produce pHBA from p-cresol. Similarly, commonly owned WO 9856920 teaches a method for the production of pHBA from toluene using a Pseudomonas mendocina mutant lacking the ability to express para-hydroxybenzoate hydroxylase (pHBH). Finally, U.S. Pat. No. 6,030,819 teaches the production of pHBA in genetically engineered E. coli expressing the chorismate pyruvate lyase (CPL) gene.
In spite of these successes the ability to produce commercially useful quantities of pHBA in microbial platforms is hampered by the use of toxic starting materials and limited biomass. A method for pHBA production that overcomes these problems is needed.
Coincidentally, pHBA is naturally occurring in nearly all plants, animals, and, microorganisms, albeit in miniscule quantities. In many bacteria, the generation of pHBA occurs by way of chorismate, an important branchpoint intermediate in the synthesis of numerous aromatic compounds, including phenylalanine, tyrosine, p-aminobenzoic acid, and ubiquinone. In E. coli, chorismate itself undergoes five different enzymatic reactions to yield five different products, and the enzyme that is ultimately responsible for the synthesis of pHBA is chorismate pyruvate lyase, which is known as CPL. The latter is the product of the E. coli ubiC gene, which was independently cloned by two different groups (Siebert et al., FEBS Lett 307:347-350 (1992); Nichlols et al., J. Bacteriol 174:5309-5316 (1992)). The enzyme is a 19 kDa monomeric protein with no known co-factors or energy requirements. Through elimination of the C3 enolpyruvyl side chain of its sole substrate, CPL catalyzes the direct conversion of 1 mol of chorismate to 1 mol of pyruvate and 1 mol of pHBA. Recombinant CPL has been overexpressed in E. coli, purified to homogeneity, and partially characterized both biochemically and kinetically (Siebert et al., Microbiology 140:897-904; Nichlols et al., J. Bacteriol 174:5309-5316 (1992)). In addition a detailed mechanism for the CPL enzyme reaction has also been proposed (Walsh et al., Chem Rev. 90:1105-1129).
In plants pHBA has been found in carrot tissue (Schnitzler et al., Planta, 188, 594, (1992)), in a variety of grasses and crop plants (Lydon et al., (J. Agric. Food. Chem., 36, 813, (1988), in the lignin of poplar trees (Terashima et al., Phytochemistry, 14, 1991, (1972); and in a number of other plant tissues (Billek et al., Oesterr. Chem., 67, 401, (1966). The fact that plants possess all of the necessary enzymatic machinery to synthesize pHBA suggests that they may be a useful platform for the production of this monomer. For example, as a renewable resource a plant platform would require far less energy and material consumption than either petrochemical or microbial methods. Similarly, a plant platform represents a far greater available biomass for monomer production than a microbial system. Finally, the natural presence of pHBA in plants suggests that host toxicity as a result of overproduction of the compound might not be a problem. Nevertheless, in spite of the obvious benefits of using plants as a means to produce pHBA, high level production of the monomer has been elusive.
One difficulty to be overcome lies in the metabolic fate of chorismate in plant tissues. Indeed, the production of pHBA from chorismate is vastly more complicated in higher plants than microbes, since the former lack an enzyme that is functionally equivalent to CPL. For example, the biosynthetic pathway leading to pHBA in Lithospermum erythrorhizon is thought to consist of up to 10 successive reactions (Loscher and Heide, Plant Physiol. 106:271-279 (1992)), presumably all catalyzed by different enzymes. Moreover, most of the enzymes that catalyze these reactions have not been identified, nor have their genes been cloned. Even less information is available on how pHBA is synthesized in other plant species. To further complicate matters, those enzymes that are known to participate in plant pHBA production span two different pathways, that are differentially regulated and located in different cellular compartments. Thus, chorismate is an intermediate of the shikimate pathway which is largely confined to chloroplasts and other types of plastids (Siebert et al., Plant Physiol. 112:811-819 (1996)) Sommer et al., Plant Cell Physiol. 39(11): 1240-1244 (1998)), while all of the intermediates downstream from phenylalanine belong to the phenylpropanoid pathway which takes place in both the cytosol and endoplasmic reticulum.
Despite the lack of understanding of how plants normally synthesize pHBA and the enzymes that are involved in this process, transgenic plants that accumulate significantly higher levels of pHBA than wildtype plants have been described. For example, Kazufumi Yazaki, (Baiosaiensu to Indasutori (1998), 56(9), 621-622) discusses the introduction of the CPL encoding gene into tobacco for the production of pHBA in amounts sufficient to confer insect resistance. Similarly, Siebert et al., (Plant Physiol. 112:811-819 (1996)) have demonstrated that tobacco plants (Nicotiana tabacum) transformed with a constitutively expressed chloroplast-targeted version of E. coli CPL (referred to as “TP-UbiC”) have elevated levels of pHBA that are at least three orders of magnitude greater than wildtype plants (WO 96/00788 granting as DE 4423022). Interestingly, the genetically modified tobacco plants contained only trace amounts of free pHBA. Instead, virtually all of the compound (˜98%) was converted to two glucose conjugates, a phenolic glucoside and an ester glucoside, that were present in a ratio of about 3:1 (Siebert et al., Plant Physiol. 112:811-819 (1996); Li et al., Plant Cell Physiol. 38(7):844-850 (1997)). Both glucose conjugates were 1-β-D-glucosides, with a single glucose residue covalently attached to the hydroxyl or carboxyl group of pHBA. The best transgenic plant that was identified in this study had a total pHBA glucoside content of ˜0.52% of dry weight, when leaf tissue was analyzed. Correcting for the associated glucose residue, the actual amount of pHBA that was produced in the transgenic tobacco plants was only about half of this value.
In more recent studies, the same artificial fusion protein was expressed in transformed tobacco cell cultures using both a constitutive promoter (Sommer et al., Plant Cell Physiol. 39(11):1240-1244 (1998)) and an inducible promoter (Sommer et al., Plant Cell Reports17:891-896 (1998)). While the accumulation of pHBA glucosides was slightly higher than the original study with whole plants, in neither case did the levels exceed 0.7% of dry weight. In contrast, when TP-UbiC was examined in hairy root cultures of Lithospermum erythrorhizon (Sommer et al., Plant Molecular Biology 39:683-693 (1999)) the pHBA glucoside content reached levels as high as 0.8% of dry weight, after correcting for the endogenous levels in the untransformed control cultures.
Although these studies demonstrate the feasibilility of using genetic engineering to increase the level of pHBA in higher plants, the TP-UbiC artificial fusion protein described above is unable to generate the compound in commercially useful quantities. Such an effort will require increasing the pHBA content of an agronomically suitable plant to levels that are 10- to 20-fold higher than those previously reported. Thus, one or more modifications of the present systems are needed to achieve these levels. Since chorismate, the substrate for CPL, is synthesised in plastids, one potential area for improvement may lie in the design of a better chloroplast targeting sequence to achieve higher levels of enzyme activity in the cellular compartment of interest. Indeed, that there is a positive correlation between CPL enzyme activity and accumulation of pHBA glucosides is apparent in several of the studies noted above (Siebert et al., Plant Physiol. 112:811-819 (1996); Sommer et al., Plant Cell Physiol. 39(11):1240-1244 (1998); Sommer et al Plant Cell Reports 17:891-896 (1998)). Furthermore, in none of these studies is there any evidence to suggest that the systems were saturated with CPL enzyme activity using the TP-UbiC artificial fusion protein.
It is well known that most naturally occurring chloroplast proteins are nuclear-encoded and synthesized as larger molecular weight precursors with a cleavable N-terminal polypeptide extension called a transit peptide. It is also generally accepted that the latter contains all of the information that is necessary for translocation into the chloroplast. Although the mechanistic details of protein import remain to be elucidated, several important facts have emerged:    (a) precursor uptake occurs post-translationally (Chua and Schmidt, Proc Natl. Acad. Sci. 75:6110-6114 (1978); Highfield and Ellis, Nature 271:420-424 (1978)) and is mediated by proteinacious receptors that exist in the chloroplast envelope membranes (Cline et al., J. Biol. Chem. 260:3691-3696 (1985)));    (b) ATP-hydrolysis is the sole driving force for translocation (Grossman et al., Nature 285:625-628 (1980); Cline et al., J. Biol. Chem. 260:3691-3696 (1985));    (c) fusion of a transit peptide to a foreign protein is at times, but not always, sufficient to trigger uptake into chloroplasts, both in vivo ((Van den Broeck et al., Nature 313:358-362 (1985)); Schreier et al., EMBO J. 4:25-32 (1985)) and in vitro Wasmann et al., Mol. Gen. Genet. 205:446-453 (1986)); and finally,  (d) following chloroplast import, the transit peptide is proteolytically removed from the precursor protein to give rise to the “mature” polypeptide. Although the complete sequence of thousands of transit peptides are now known, the manipulation of these sequences to achieve optimal targeting and expression of foreign proteins in the chloroplast compartment of plants is still a matter of trial and error. It is well settled however, that simply attaching a transit peptide to a foreign protein does not necessarily guarantee that it will be efficiently taken up by chloroplasts or properly processed. Even when the same targeting sequence is fused to different proteins, the results are completely unpredictable (Lubben et al., The Plant Cell 1: 1223-1230 (1989)), and the different passenger proteins are transported with different efficiencies. The reasons for this are not clear, however it has been suggested that chloroplast uptake and removal of the transit peptide are somehow coupled, and that certain artificial fusion proteins are either not processed or processed ineffectively. For example, it has been shown that even very subtle changes in the vicinity of the natural cleavage site of the Rubisco small subunit precursor can lead to aberrant processing (Robinson and Ellis, Eur. J. Biochem. 142:342-346 (1984); Robinson and Ellis, Eur. J. Biochem. 152:67-73 (1985)) and diminished chloroplast uptake (Wasmann et al., J. Biol. Chem. 263:617-619 (1988)).
Some degree of improvement has been achieved in this area by including in the chloroplast targeting sequence not only the transit peptide and the scissile bond, but also a small portion of the mature N-terminus of the transit peptide donor. Indeed, this approach has worked both in vivo and in vitro ((Van den Broeck et al., Nature 313:358-362 (1985); Schreier et al., EMBO J. 4:25-32 (1985); Wasmann et al., Mol. Gen. Genet. 205:446-453 (1986); Herrera-Estrella et al., EP 0189707; U.S. Pat. No. 5,728,925; U.S. Pat. No. 5,717,084) for another bacterial protein, namely, neomycin phosphotransferase II (NPT-II). Thus, a chimeric protein consisting of the transit peptide of the Rubisco small subunit precursor plus the first 22 residues of mature Rubisco fused to the N-terminus of NPT-II was taken up by chloroplasts much better than a similar construct that only contained the transit peptide and scissile bond. This strategy is not foolproof however, and is still associated with a high degree of unpredictability that is inextricably linked to the passenger protein. This is most readily seen in the literature attempts to target CPL to chloroplasts. For example Sommer et al., Plant Cell Physiol. 39(11):1240-1244 (1998)) describes an analogous artificial fusion protein comprising the CPL gene product fused at its N-terminus to the transit peptide and first 21 amino acid residues of the Rubisco small subunit (e.g., “TP21UbiC”). While it was anticipated that this modification would improve chloroplast uptake and processing, the cells that contained the original construct, TP-UbiC, had much higher levels of both CPL enzyme activity and pHBA glucosides. Thus, application of the teaching of Wasmann et al., (Mol. Gen. Genet. 205:446-453 (1986)) had a detrimental effect on a different protein.
The problem to be solved therefore is to provide a method for the production of pHBA in plants at commercially useful levels taking advantage of the chemical reaction that is catalyzed by the bacterial protein CPL. This is a particularly ambitious goal since on top of all of the complications noted above it is clear from the literature that certain N-terminal modifications of E. coli CPL can result in a substantial loss of enzyme activity (Siebert et al., Plant Physiol. 112:811-819 (1996). Consequently, it is not only essential to identify an artificial fusion protein that is efficiently imported into chloroplasts, but one that is also proteolytically processed to yield either unmodified CPL or a CPL variant with an N-terminal extension that doesn't interfere with enzyme activity. The solution to this problem is not taught in the art. Applicant has solved the stated problem by creating a novel artificial fusion protein that enables the expression of sufficiently high levels of CPL enzyme activity in chloroplasts to accumulate commercially useful levels of pHBA.